The regulation of fat storage has considerate implications for metabolic disease, obesity, and even cancer. Fatty acids are obtained directly from absorption of dietary fats, or from conversion of digested nutrients to fatty acids via de novo synthesis. We developed a 13C isotope-labeling assay to quantify fatty acid synthesis in nematodes and applied this strategy to discover regulators of fatty acid synthesis via C. elegans genetics. The objective of this application is to more thoroughly characterize two novel mechanisms identified in our screen. First, we will investigate a type II PI3K called HLS-1, little is known about the metabolic role of type II PI3Ks in worms or mammals. Our preliminary data show that HLS-1 is a strong regulator of lipogenesis in C. elegans, and may do so by participating in a previously uncharacterized branch of the insulin signaling pathway. We will directly test this hypothesis and more thoroughly establish the downstream regulatory effects of HLS-1 on lipid metabolism. Second, we uncovered an intriguing relationship between oxidative phosphorylation and fatty acid synthesis. In particular, inhibition of mitochondrial respiration leads to strong elevation of fatty acid synthesis and lipid storage. This observation not only has implications for metabolic disease, but may also explain the critical role of fatty acid synthesis in cancer. The second objective of this proposal is to exploit genetic and biochemical approaches to elucidate the mechanism by which mitochondrial respiration impacts lipogenesis, testing in particular, a model whereby fatty acid synthesis maintains redox balance in hypoxic cancer cell environments. PUBLIC HEALTH RELEVANCE: Fat storage is determined by a balance between dietary fat intake, fat synthesis, and fat expenditure. Consequently, regulators of any of these processes are likely to be of significant interest to metabolic disease and obesity. We have exploited the genetic advantages of C. elegans to identify completely novel regulators of fatty acid synthesis, regulators that could serve as important targets for treating obesity and metabolic disease. In this application, we will carry out experiments to more thoroughly characterize how these novel regulators impart their effects on fat synthesis and storage.